Porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and cultivation method thereof

A technology of respiratory syndrome and breeding method, applied in the field of porcine reproductive and respiratory syndrome virus receptor CD163 knockout pigs

Inactive Publication Date: 2012-10-10
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Transgenic animals with CD163 gene knockout have not been reported yet

Method used

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  • Porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and cultivation method thereof
  • Porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and cultivation method thereof
  • Porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and cultivation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Construction of PRRSV Receptor CD163 Gene Knockout Vector

[0057] 1 Plasmid The important intermediate plasmids formed are pGM-T-Sarm-CD163 and pGM-T-Larm-CD163.

[0058]2 Experimental process Using the miniature pig genome as a template, the PCR method (see Table 2) was used to amplify the homologous left arm and right arm of the cloned pig CD163 respectively; -T were connected to construct pGM-T-Sarm-CD163 and pGM-T-Larm-CD163 respectively; pGM-T-Sarm-CD163 was digested with BamHI and hindIII, and the homologous right arm Sarm fragment of CD163 gene was recovered; Digest pGM-T-Larm-CD163, recover CD163 gene homologous left arm Larm fragment; CD163 gene homologous left arm and homologous right arm were respectively combined with positive selection gene Bip-Neo and negative selection gene HSV-t The targeting vector pSSC-9 was connected to construct a CD163 gene knockout vector containing positive and negative selection markers (see attached figure 1 ). The PCR pr...

Embodiment 2

[0066] Construction of porcine embryonic fibroblasts with CD163 gene knockout

[0067] 1 Plasmid pSSC-Larm-Sarm-CD163

[0068] 2-cell minipig embryonic fibroblasts

[0069] 3 Experimental process

[0070] 3.1 Vector linearization: extract pSSC-Larm-Sarm-CD163 plasmid, use Sfi I digestion, followed by ethanol precipitation, with sterile ddH 2 O dissolved.

[0071] 3.2 Cell transfection: The linearized vector was transfected into minipig fibroblasts according to the instructions of liposome Fugene HD (Invitrogen), and the cells were incubated in 5% CO 2 , cultured at 39°C.

[0072] The day before the transfection, the primary cultured embryonic fibroblasts were resuscitated into a 60mm dish with antibiotic-free culture medium, and the cells could be transfected when the cells reached 70-80% confluency. 24 h after transfection, the cells were passaged by trypsinization at 1:21. After 48 h of transfection, G418 selection medium (300 μg / ml concentration) was added. When...

Embodiment 3

[0090] Generation of gene knockout pigs by somatic cell nuclear transfer

[0091] 1 cell CD163 knockout porcine embryonic fibroblasts were constructed. the

[0092] 2 Animals Healthy Duroc sows (red), Landrace pigs (white).

[0093] 3. Experimental process Somatic cell nuclear transfer technology was used to construct CD163 gene knockout pigs.

[0094] 3.1 Collect pig ovaries from the slaughterhouse, place them in a thermos bottle containing double-antibody saline at 25-37 °C, and return them to the laboratory within 2 hours. The ovaries were washed several times with 37°C normal saline containing double antibodies, and the ovaries were placed in a 37°C water bath. Use a 20 mL syringe with a 18-gauge needle to aspirate 2-8 mm follicles on the ovary, and use a needle to pick out cumulus-oocytes with uniform cytoplasm and more than two layers of dense cumulus cells under a solid microscope. Cell complexes (COCs), after maturation in maturation medium, were removed by blind...

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Abstract

The invention provides a porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and a cultivation method thereof. A constructed CD163 gene knock-out carrier pSSC-Larm-Sarm-CD163 uses a genome of Chinese experimental miniature swines as a template, and adopts a PCR method to amplify a homologous left arm and a homologous right arm of the swine CD163 gene; the cloned homologous left arm and the homologous right arm of the CD163 gene are subcloned to a pSSC-9 carrier respectively; and the porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine is cultivated, wherein the CD163 gene is not expressed, and the swine is not infected by the porcine reproductive and respiratory syndrome virus.

Description

Technical field: [0001] The invention provides a porcine reproductive and respiratory syndrome virus receptor CD163 knockout pig, and also discloses a breeding method of the pig, which belongs to the technical field of bioengineering. Background technique: [0002] Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV) and is characterized by abortion, stillbirth, fetal mummification, and respiratory disease infectious disease. During the course of the disease, there will be transient skin cyanosis in both ears, so it is also called "blue ear disease". In recent years, the disease has shown an obvious high incidence trend in China, which has caused great losses to the pig industry and has become one of the important infectious diseases that seriously threaten the development of my country's pig industry. Fully differentiated primary porcine alveolar macrophages are the target cells for PRRSV infection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/63C12N5/10
Inventor 赖良学逄大欣欧阳红生任林柱宋娜于飞飞杨鑫陈福旺曹宇航李莉
Owner JILIN UNIV
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